Hemolysis intraoperative monitoring by Helge V-test in patients undergoing extracorporeal circulation in cardiac surgery: a single center data

Hemolysis intraoperative monitoring by Helge V-test in patients undergoing extracorporeal circulation in cardiac surgery: a single center data | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Hemolysis intraoperative monitoring by Helge V-test in patients undergoing extracorporeal circulation in cardiac surgery: a single center data Lorenzo Schiavoni, Mariapia Stifano, Alessia Mattei, Francesca La Verde, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4632556/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Hemolysis is a complication in all surgical procedures requiring extracorporeal circulation (ECC). The aim of this study is to evaluate the effectiveness of the disposable point of care device Hemcheck Helge™ V-TEST quantifying hemolysis during cardiac surgery in ECC. Methods Two whole blood samples of 78 patients were simultaneously collected at different times of surgery: T0 pre ECC; T1 at the time of clamping of aorta; T2 at 20 minutes after the start of ECC; T3 at the end of ECC; T4 at the end of surgery. Both samples were analyzed by the disposable Hemcheck Helge™ V-TEST device, which offers a real-time assessment of hemolysis through a value of plasma free hemoglobin (PfHb) expressed in mg/dL. Results Out of all cases analyzed, no case of hemolysis (PfHb ≥ 50 mg/dL) was recorded at T0. The results recorded median PfHb values at T0 0.5 (0-7.1) mg/dL, at T1 3.75 (0-14.4) mg/dL; at T2 8.25 (0.4–19.1) mg/dL, at T3 27.5 (9.9–50) mg/dL, at T4 18.5 (2.4–41) mg/dL; for all T time p value was 50 mg / dL at T3 and ECC time > 100 minutes (p < 0,05). Conclusions The use of Hemcheck Helge™ V-TEST allows effective identification of hemolysis directly in the operating room, reducing wasted time for laboratory analyses. This could help the anesthetist, perfusionist or cardiac surgeon approaching earlier intraoperative hemolysis and its effects on organs function and improve the post-operative course of patients undergoing cardiac surgery in ECC. Figures Figure 1 INTRODUCTION Red blood cells (RBCs) are bi-concave discoid cells without a nucleus that perform the oxygen transport from lungs to tissue for cell respiration and then energy production via oxidative phosphorylation. Their shape, viscosity and viscoelasticity of their cytoskeleton allow the red blood cells to deform and adapt to different calibers of the blood vessels to reach all tissues and cells. (1) Oxygen transport is possible by binding with high concentrations of hemoglobin present in the cytosol of erythrocytes. Hemoglobin is a tetrameric protein made up of two alpha and two beta subunits, each containing a ferrous iron capable of binding an oxygen molecule. The average lifespan of red blood cells in the human blood circulation is 120 days. Several strategies protect RBCs from oxidative stress due to high concentrations of iron and oxygen, until older RBCs are removed from the bloodstream by reticuloendothelial system macrophages in the liver and spleen. (2) During cardiac surgery requiring ECC, the passage of blood through the rollers, as well as exposure to non-endothelial surfaces at different speeds and blood-air interface, causes mechanical damage to erythrocytes proportional to the level of shear stress and exposure time. (3) RBCs lysis releases cell content into the plasma. Various scavenging mechanisms avoid free hemoglobin and heme free radicals’ toxic effects. Free hemoglobin is irreversibly bound by circulating haptoglobin, produced by the liver. Hemoglobin-haptoglobin complexes are then rapidly cleared from the circulation via monocytes and tissue macrophages. (4) However, excessive hemolysis determines the saturation of the intravascular scavenging mechanisms, with an increase in the blood content of PfHb and the heme group with consequent iron overload and nitric oxide (NO) depletion. In fact, PfHb binds endothelial NO irreversibly, causing increased systemic vascular resistance (SVR), elevated pulmonary vascular resistance (PVR), increased thrombin formation, fibrin deposition, platelet activation and aggregation and, finally, organ dysfunction. (5) PfHb also appears to impair left ventricular function and coronary flow, as shown by a study conducted by Nemoto and colleagues on neonatal rabbits, in which the effects of PfHb were much more evident in cases of ischemia and reperfusion. (6) Furthermore, iron overload has been associated with an increased risk of acute renal failure and lung permeability. Much more, bilirubin storage has been associated with a high mortality rate. In addition, red blood cell membrane can also be damaged. Red blood cells lose their characteristic deformability which allows them to enter the microcirculation and, ultimately, limits oxygen supply. Likewise, effective gas exchange in the pulmonary microcirculation is reduced. Lack of compensatory mechanisms such as NO-induced vasodilation can lead to cellular ischemia and ultimately organ damage. (5) The aim of this study is to evaluate the efficacy and safety of the disposable device Hemcheck Helge™ V-TEST on the assessment and quantification of the degree of hemolysis in patients undergoing ECC during Cardiac Surgery, in order to prevent and predict serious complications. METERIALS AND METHODS ETHICAL APPROVAL The study was approved by the Ethics Committee of the Fondazione Policlinico Universitario Campus Bio Medico of Rome with protocol 35.22 OSS dated 05/24/2022. HELGE TM TEST Helge TM Test (HemCheck Sweden AB) is a point of care (POC) disposable product intended for the identification of hemolyzed blood. There are three different disposable tests: s-Test and bgs-Test for blood samples in blood gas syringes and V-Test, that we used, for blood samples in vacuum tubes. The product is compatible with vacuum tubes such as heparin tubes, citrate tubes, EDTA tubes and serum tubes, except serum tubes with thrombin as an additive and required only a small amount of blood. Helge TM Test must be used in combination with a reader called “Helge H10 Reader”. The color of the plasma/serum is analyzed photometrically by the reader's integrated camera to determine the amount of free hemoglobin. The result presented by the Helge H10 reader is binary and is communicated through a red or green light. An accessory screen, Zafena POC-Workstation (HemCheck Sweden AB), with integrated printer and barcode reader, was connected to the system to enable extraction of quantified free hemoglobin values, expressed in in mg/dL, g/L o μmol/L. The aim is to find hemolyzed blood samples in a few seconds in connection with the blood-sampling or quantifying the amount of free hemoglobin in the blood. The innovative aspect is the detection of hemolysis in whole blood in REAL TIME, helping to ensure the quality of samples and to make immediate decisions. A study at Swedish emergency department showed that a cut-off of 50 mg/ml PfHb has a sensitivity of 80%, specificity of 99%; positive and negative predictive values was 89% and 98% respectively. (7) The binary result of the reader is set with this cut off, but it could be changed. STUDY DESIGN The primary objective of this study was to demonstrate Helge TM V-Test effectiveness and safety in the early identification of hemolysis in patients undergoing elective cardiac surgery in ECC. Inclusion criteria were: indication for elective cardiac surgery in ECC, age ≥ 18 years, ASA I-IV, informed consent approval; exclusion criteria were: not-approval informed consent, age < 18 years, ASA IVE, weight < 30 kg. All surgery were conducted under general anesthesia and ultrasound-guided bilateral parasternal block, with complete monitoring of vital parameters. Blood samples were taken during the surgery at different times: T0: pre-ECC, T1: at aortic clamping, T2: after 20 minutes since the ECC started, T3: at the end of ECC, T4: at the end of surgery. All blood samples were performed from arterial access. In order to avoid handling errors and variations, a double sampling was carried out and both was tested. The blood samples were then analyzed using the Helge TM V-TEST disposable device by appropriately trained specialist anesthetists and residents. A total of ten test per patients were executed and collected. The cut-off to define the presence or absence of hemolysis was maintained at 50 mg/dL, as set by the manufacturer. (7) Furthermore, patients' personal and anthropometric data were collected, as well as the pre- and post-operative hematocrit, the type of cannulas used by the perfusionists, the duration of the ECC and the clamping, the possible performance of pre-operative bloodletting and, finally, the use of vasoconstrictors. STATISTICAL METHODS All collected data were analyzed using the IBM SPSS Statistics editor and expressed in medians ± interquartile ranges. The data obtained from the two blood samples analyzed for each T were considered as the average of the two values (± standard deviation). The 1-Sample Wilcoxon test and the independent-samples Mann-Whitney test were performed on all T time values. The Spearman Rank test was used as a correlation test. Binary logistic regression was finally conducted to analyze the relationship between ECC and aortic clamping times and PfHb values at T1, T2 and T3 based on the data obtained from the rank test. P value was defined as < 0.05. The PfHb value is expressed in mg/dL. Results Since January 2022 to March 2023, 100 consecutive patients undergoing elective cardiac surgery in ECC were enrolled in the study. Informed consent was obtained from all of them. 18 were excluded for ASA IVE class risk and 4 were lost during data collecting. 78 patients were finally analyzed. ( Table 1 ) Of these 78 patients, the median age was 71 (IR 33-89) years, 63 patients enrolled were women while 15 were men. The median body surface area (BSA) was 1.89 (IR 1,4-2,3) m 2 . The median preoperative hematocrit (HCT) was 40 (IR 25-51) % and the post-operative HCT was 30 (IR 22-42) %. Finally, the median duration of CEC was 70 (IR 30-193) min, while that of aortic clamping was 56 (IR 23-133). The median size of cannulae used in CEC was 32 (IR 15-40) Fr for arterials and 40 (IR 19-46) for venous one. ( Table 2 ) Out of all cases analyzed, no case of hemolysis (PfHb value ≥ 50 mg/dL) was recorded at T0. The frequency of hemolysis at T1 was 6.4% (5 of 78), at T2 was 5.1% (4 of 78), at T3 was 24.4% (19 of 78) and at T4 was 16.7% (13 of 78). The results recorded median PfHb values at T0 0.5 (IR 0-7.1) mg/dL, at T1 3.75 (IR 0-14.4) mg/dL; at T2 8.25 (IR 0.4-19.1) mg/dL, at T3 27.5 (IR 9.9-50) mg/dL, at T4 18.5 (IR 2.4-41) mg/dL; for all T time p value was 50 mg/dL in all T times N. Absolute value; % Percentage value Table 4 PfHb value in mg/dL in all T times A statistically significant correlation (p value < 0.001) was also found between the duration of the ECC and the PfHb value at T3 (end of ECC) and T4 (end of surgery). Likewise, the duration of aortic clamping was significantly correlated with the PfHb value at T3 and T4. Instead, the PfHb value at all T time points was not related to patient age, gender, preoperative HCT value and ECC cannula diameter. Hemolysis occurred for a minimum ECC time of 35 minutes and a minimum clamp time of 31 minutes Finally, our statistical analysis did not demonstrate a significant difference between PfHb values at T1 and at T2 (p = 0.130), a statistically significant difference was instead found between PfHb at all the other T value, particularly among PfHb values at T1-T2 and at T3 (p < 0.001). Complete data are shown in the table. ( Table 5 ) Based on these data, we conducted a binary logistic regression analysis to correlate the occurrence of hemolysis in T1, T2, and T3 and the duration of both ECC and aortic clamping. These analyses demonstrated that there is no correlation between the duration of ECC and PfHB > 50 mg/dL in T1 and T2, whereas in T3 the probability of hemolysis increases for a ECC duration greater than 100 minutes. Similarly, the duration of aortic clamping does not influence the PfHb value at T1 and T2, but in T3 these increase if the clamping time is greater than 84 minutes. ( Figure 1 ) Discussion Hemolysis in all extracorporeal circuits is a matter of fact. Three distinct mechanisms are involved in hemolysis: natural selection in the spleen; physical or chemical imbalances, mechanical trauma. (8) The use of ECC in cardiac surgery determines the destruction of red blood cells throughout different pathways, with an increase in PfHb and a reduction in haptoglobin levels in the blood circulation. (3) In 2009, Pohlman and colleagues conducted a study on sheep and human blood samples reporting how the negative pressure exerted by the pump, together with the exposure of the blood to air, increased the degree of hemolysis. (9) Five years earlier Kameneva et al. reported how the intensity of hemolysis depends on the aspiration flow rate, the hematocrit, the duration of the ECC and the storage time of the concentrated red blood cells used during the CPB procedure. (10) Other authors suggest that the onset of hemolysis and inflammation also depends on the duration and conduction of CPB and on the material used in that process. (11) The release of free constituents of red blood cells and the consumption of scavenger mechanisms, as well as hemorheological alterations, lead to important clinical alterations that can promote organ failure. (5) The mechanisms of cellular mechanical damage during CPB have been described by several investigators: positive pressure, wall impact forces, nonendothelial blood surfaces, negative pressure, blood-air interface, and shear stress have all been identified as responsible forces. (5) It is known that the extracorporeal circuit is made up of two different types of pumps: roller or centrifugal. If roller pumps force the blood flow inside a tube which is pinched by rotating rollers, centrifugal pumps move the flow by generating kinetic energy from the rotation of an impeller. (12) The type of pump used to perform CPB can influence hemolysis; specifically, roller pumps are associated with a greater amount of hemolysis than centrifugal pumps. (13) Bhirowo and colleagues conducted a meta-analysis of 64 RCTs from 1990 to 2021 involving a total of 3384 patients undergoing various cardiac surgeries. According to the authors, centrifugal pumps are associated with a significant reduction in hemolysis in terms of PfHb and HP levels compared to roller pumps (RP), but not in LDH levels. As in our trial, higher serum PfHb concentrations were reported in groups undergoing longer-duration ECC. Furthermore, shear stress due to air aspiration via cardiotomy is considered another of the main cause of hemolysis during CPB. Significant reductions in hemolysis levels were observed in gravity-assisted venous drainage (GAVD) versus vacuum-assisted venous drainage (VAVD), continuous flow versus pulsatile flow, and using propofol versus isoflurane 1-2 %. (14) Our study aims to demonstrate the effectiveness, speed and safety of the Helge TM -V-TEST device in identifying hemolysis in patients undergoing these interventions. Our statistical analysis demonstrated a significant difference between PfHb values in T3 (end of ECC) and in T1-T2. The same difference is not present between T1 (aortic clamping) and T2 (20' after ECC started), therefore we can deduce that the only factor determining hemolysis is the duration of the ECC and the aortic clamping, given the same patient-related factors. In particular, the degree of hemolysis increases for ECCs exceeding 100 minutes. Statistically significant differences were also found between T1-T2 and T0 (pre-ECC); however, the onset of ECC is an independent and non-modifiable factor for this type of surgery, unlike the duration. Therefore, we can identify the cut off of 100 minutes of ECC beyond which it becomes justifiable to evaluate the degree of hemolysis with the Helge TM -V-TEST system to make intraoperative adjustments, in order to reduce the degree of hemolysis and its clinical consequences. For example, in patients in whom long-lasting ECC is expected it could be useful, as suggested by Vercaemst et al., the use of wider and shorter connecting tubes, avoiding angled bends, to minimize the gradients necessary to push the blood along the tube and the transition from laminar flow to turbulent flow. (5) At the same time, an increase in pfHb during ECC above the critical value of 50 mg/dL allows for the implementation of preventive strategies to avois clinically evident hemolysis, such as a reduction in hematocrit through hemodilution implemented directly by the perfusionist via CBP or, for adequate DO2 values, a reduction in ECC flows. Another strategy could be to switch from pulsed flow to continuous flow. Similarly, drugs such as pentoxifylin (PTX), polyethylene glycol (PEG) molecules, melatonin, simvastatin or inhaled NO (iNO), may have some benefits on hemolysis. (5) Passaroni et al. studied the correlations between hemolysis and inflammation after ECC, with increased risk of organ dysfunction and multisystem complications, which could have a critical role on the duration of mechanical ventilation and post-operative stay in intensive care. Acute renal failure (AKI) is a frequent complication after cardiac surgery in CPB and in 1-5% requires post-operative hemodialysis, with a significant increase in mortality and morbidity. (15) The causes are different and not all treatable, including age, anemia, diabetes, chronic lung disease, chronic heart failure, chronic renal dysfunction, administration of nephrotoxic agents, hypoperfusion, hypotension, embolization and increased aortic clamping time. A frequent cause is hemolysis. Hokka et al. conducted a prospective observational study on 74 patients with chronic renal failure who underwent cardiac surgery on the valve and aortic arch in ECC from 2014 to 2020, to evaluate the association between perioperative levels of free hemoglobin and haptoglobin and acute renal failure. AKI occurred in 25 patients (33.8%) and their multivariable analysis demonstrated an independent increase in risk with maximum PfHb values and minimum haptoglobin values. The independent association of increased PfHb values with the risk of AKI occurred specifically one hour after the start of CPB. The authors suggest the use of filters to remove PfHb or the administration of exogenous haptoglobin or epicidin for the prevention of AKI in case of serum PfHb ≥ 0.06 g/dL one hour after starting CPB or ≥ 0 .12 g/dL at any time. (16) Another study evaluated the impact of hemolysis in cardiac surgery patients on plasma NO consumption, AKI and intestinal tissue damage after cardiac surgery. Likewise, in patients undergoing CPB, an increase in plasma PfHb levels and a reduction in plasma haptoglobin and, not surprisingly, in NO levels were recorded, in a manner proportional to the duration of ECC. These data were associated with an increase in urinary N-acetyl-b-D-glucosaminidase (NAG) and plasma intestinal fatty acid binding protein (IFABP), concluding that hemolysis plays an important role in postoperative renal damage and to the intestinal mucosa, as well as direct cytotoxic damage potentially limiting the bioavailability of NO. It is obvious that this has important consequences on the post-operative course, increasing the risk of AKI, but also of systemic inflammatory response (SIRS) or sepsis. (17) All studies analyzed assessed hemolysis by measuring levels of PfHb, haptoglobin, LDH or by calculating the hemolysis index. (18) A wide range of first- and second-line laboratory tests are currently available in the diagnostic approach to hemolysis but none of them can be performed bedside. Erythrocytes also contain the enzyme LDH, whose serum levels increase in case of hemolysis, but it cannot be considered a specific marker. In case of non-clinically evident hemolysis, once normocytic or, in some cases, macrocytic anemia has been identified, laboratory tests for a correct diagnosis should also include the measurement of reticulocytes and unconjugated bilirubin, which does not can no longer be eliminated properly, as can the search for blood in the urine. It is obvious that this request is an enormous waste of resources and above all of time. (19) The laboratory measurement of hemiglobincyanide is currently the gold standard, but it is associated with toxicity and does not allow a timely diagnosis, on the other hand the evaluation of haptoglobin is associated with a high rate of false negatives. This has led to the development of new clinical chemistry analyzers in laboratories, such as the HIL index (Hemoglobin, icterus, lipaemia/turbidity) which can estimate the presence of fHb, bilirubin and turbidity in samples. (7)(18) The main strength of our study is the inclusion of patients undergoing different types of cardiac surgery of varying complexity. This allowed us to evaluate the effectiveness and safety of the Helge TM -V-TEST in different operating scenarios. Having established that the development of hemolysis is directly related to the extracorporeal circuit and the duration of ECC, implementing preventive interventions could help to limit hemolysis-related damage, to correct intraoperative management in case of PfHb rising and improve the post-operative outcome of these patients. Our study, however, was limited to evaluating the feasibility, reliability, and safety of the point-of-care examination, without distinction based on the type of surgery and patient comorbidities. For example, because haptoglobin is synthesized in the liver, patients with impaired liver function may have reduced haptoglobin production which could result in increased PfHb independent of red blood cell lysis. (5) From a future perspective, it could be interesting to analyze the effectiveness of the test in the various subgroups of patients. Conclusion Evaluating and quantifying hemolysis during ECC using a point-of-care system as Helge TM V-TEST allows real-time corrective strategies to be implemented to prevent hemolysis-related complications and could improve the outcome of patients undergoing elective cardiac surgery, especially for ECC duration over 100 minutes. Declarations The authors have no relevant financial or non-financial interests to disclose. L.S. conceptualization, investigation, methodology, writing-review and editing. M.S. writing-original draft, formal analysis and data curation, software. A.M. writing-review and editing, validation F.L.V. conceptualization, investigation, methodology A.S., A.L.D.P, D.S., S.R., M.C.C., L.M. data curation R.C. project administration and resources F.C., G.P. supervision A.R., E.C. conceptualization and data curation F.E.A., M.C. supervision, validation and visualization All authors read and approved the final manuscript. References Corrons JLV, Casafont LB, Frasnedo EF (2021) Concise review: how do red blood cells born, live, and die? Ann Hematol. 100(10):2425-2433. https://doi.org/10.1007/s00277-021-04575-z Yoshida T, Prudent M, D'Alessandro A (2019) Red blood cell storage lesion: causes and potential clinical consequences. 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(1999) Acute renal failure following cardiac surgery. Nephrol Dial Transplant. 14(5):1158-62. https://doi.org/10.1093/ndt/14.5.1158. Hokka M, Egi M, Kubota K, Mizobuchi S. (2021) Perioperative Serum Free Hemoglobin and Haptoglobin Levels in Valvular and Aortic Surgery With Cardiopulmonary Bypass: Their Associations With Postoperative Kidney Injury. J Cardiothorac Vasc Anesth. 35(11):3207-3214. https://doi.org/10.1053/j.jvca.2021.04.029. Vermeulen Windsant IC, de Wit NC, Sertorio JT, van Bijnen AA, Ganushchak YM, Heijmans JH, Tanus-Santos JE, Jacobs MJ, Maessen JG, Buurman WA. (2014) Hemolysis during cardiac surgery is associated with increased intravascular nitric oxide consumption and perioperative kidney and intestinal tissue damage. Front Physiol. 5:340. https://doi.org/10.3389/fphys.2014.00340. Lippi G, Favaloro EJ, Franchini M. Haemolysis index for the screening of intravascular haemolysis: a novel diagnostic opportunity?(2018) Blood Transfus. 16(5):433-437. https://doi.org/10.2450/2018.0045-18. Phillips J, Henderson AC. (2018) Hemolytic Anemia: Evaluation and Differential Diagnosis. Am Fam Physician. 98(6):354-361. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4632556","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":322508484,"identity":"965b88b9-6763-4520-833b-01532c16187b","order_by":0,"name":"Lorenzo Schiavoni","email":"","orcid":"","institution":"Fondazione Policlinico Universitario Campus Bio- Medico","correspondingAuthor":false,"prefix":"","firstName":"Lorenzo","middleName":"","lastName":"Schiavoni","suffix":""},{"id":322508485,"identity":"331d0f57-5b7f-4af5-99cd-628e705282b9","order_by":1,"name":"Mariapia 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Bio-Medico","correspondingAuthor":false,"prefix":"","firstName":"Elena","middleName":"","lastName":"Casali","suffix":""},{"id":322508504,"identity":"79f78c0b-69e1-47e0-bb44-e4f42bb0c601","order_by":15,"name":"Felice Eugenio Agrò","email":"","orcid":"","institution":"Fondazione Policlinico Universitario Campus Bio- Medico","correspondingAuthor":false,"prefix":"","firstName":"Felice","middleName":"Eugenio","lastName":"Agrò","suffix":""},{"id":322508505,"identity":"5b3b5545-2c85-43e3-bca7-6b37aaa666f7","order_by":16,"name":"Massimiliano Carassiti","email":"","orcid":"","institution":"Fondazione Policlinico Universitario Campus Bio- Medico","correspondingAuthor":false,"prefix":"","firstName":"Massimiliano","middleName":"","lastName":"Carassiti","suffix":""}],"badges":[],"createdAt":"2024-06-24 23:53:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4632556/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4632556/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60633419,"identity":"a4910c2f-b7b5-4918-b512-627b94a75add","added_by":"auto","created_at":"2024-07-19 01:39:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":268396,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePfHb value (mg/dL) in T3 (A-B), T1(C) and T2 (D). \u003c/strong\u003eBy\u003cstrong\u003e \u003c/strong\u003eIBM SPSS Statistics editor\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4632556/v1/42edb000e1a7195d22cd45c1.png"},{"id":62212101,"identity":"7e8f7cec-c4de-45cc-9a8f-1a8a06cbc05c","added_by":"auto","created_at":"2024-08-11 09:22:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1026408,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4632556/v1/9d3c4f33-562e-4f63-9ab1-5749c1977f8d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hemolysis intraoperative monitoring by Helge V-test in patients undergoing extracorporeal circulation in cardiac surgery: a single center data","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eRed blood cells (RBCs) are bi-concave discoid cells without a nucleus that perform the oxygen transport from lungs to tissue for cell respiration and then energy production via oxidative phosphorylation. Their shape, viscosity and viscoelasticity of their cytoskeleton allow the red blood cells to deform and adapt to different calibers of the blood vessels to reach all tissues and cells. (1)\u003c/p\u003e \u003cp\u003eOxygen transport is possible by binding with high concentrations of hemoglobin present in the cytosol of erythrocytes. Hemoglobin is a tetrameric protein made up of two alpha and two beta subunits, each containing a ferrous iron capable of binding an oxygen molecule. The average lifespan of red blood cells in the human blood circulation is 120 days. Several strategies protect RBCs from oxidative stress due to high concentrations of iron and oxygen, until older RBCs are removed from the bloodstream by reticuloendothelial system macrophages in the liver and spleen. (2)\u003c/p\u003e \u003cp\u003eDuring cardiac surgery requiring ECC, the passage of blood through the rollers, as well as exposure to non-endothelial surfaces at different speeds and blood-air interface, causes mechanical damage to erythrocytes proportional to the level of shear stress and exposure time. (3)\u003c/p\u003e \u003cp\u003eRBCs lysis releases cell content into the plasma.\u003c/p\u003e \u003cp\u003eVarious scavenging mechanisms avoid free hemoglobin and heme free radicals\u0026rsquo; toxic effects.\u003c/p\u003e \u003cp\u003eFree hemoglobin is irreversibly bound by circulating haptoglobin, produced by the liver. Hemoglobin-haptoglobin complexes are then rapidly cleared from the circulation via monocytes and tissue macrophages. (4)\u003c/p\u003e \u003cp\u003eHowever, excessive hemolysis determines the saturation of the intravascular scavenging mechanisms, with an increase in the blood content of PfHb and the heme group with consequent iron overload and nitric oxide (NO) depletion. In fact, PfHb binds endothelial NO irreversibly, causing increased systemic vascular resistance (SVR), elevated pulmonary vascular resistance (PVR), increased thrombin formation, fibrin deposition, platelet activation and aggregation and, finally, organ dysfunction. (5)\u003c/p\u003e \u003cp\u003ePfHb also appears to impair left ventricular function and coronary flow, as shown by a study conducted by Nemoto and colleagues on neonatal rabbits, in which the effects of PfHb were much more evident in cases of ischemia and reperfusion. (6)\u003c/p\u003e \u003cp\u003eFurthermore, iron overload has been associated with an increased risk of acute renal failure and lung permeability. Much more, bilirubin storage has been associated with a high mortality rate. In addition, red blood cell membrane can also be damaged. Red blood cells lose their characteristic deformability which allows them to enter the microcirculation and, ultimately, limits oxygen supply. Likewise, effective gas exchange in the pulmonary microcirculation is reduced. Lack of compensatory mechanisms such as NO-induced vasodilation can lead to cellular ischemia and ultimately organ damage. (5)\u003c/p\u003e \u003cp\u003eThe aim of this study is to evaluate the efficacy and safety of the disposable device Hemcheck Helge\u0026trade; V-TEST on the assessment and quantification of the degree of hemolysis in patients undergoing ECC during Cardiac Surgery, in order to prevent and predict serious complications.\u003c/p\u003e"},{"header":"METERIALS AND METHODS","content":"\u003ch3\u003eETHICAL APPROVAL\u003c/h3\u003e\n\u003cp\u003eThe study was approved by the Ethics Committee of the Fondazione Policlinico Universitario Campus Bio Medico of Rome with protocol 35.22 OSS dated 05/24/2022.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eHELGE\u003csup\u003eTM\u003c/sup\u003e TEST\u003c/h3\u003e\n\u003cp\u003eHelge\u003csup\u003eTM\u003c/sup\u003e Test (HemCheck Sweden AB) is a point of care (POC) disposable product intended for the identification of hemolyzed blood.\u003c/p\u003e\n\u003cp\u003eThere are three different disposable tests: s-Test and bgs-Test for blood samples in blood gas syringes and V-Test, that we used, for blood samples in vacuum tubes. The product is compatible with vacuum tubes such as heparin tubes, citrate tubes, EDTA tubes and serum tubes, except serum tubes with thrombin as an additive and required only a small amount of blood.\u003c/p\u003e\n\u003cp\u003eHelge\u003csup\u003eTM\u003c/sup\u003e Test must be used in combination with a reader called \u0026ldquo;Helge H10 Reader\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003eThe color of the plasma/serum is analyzed photometrically by the reader\u0026apos;s integrated camera to determine the amount of free hemoglobin.\u003c/p\u003e\n\u003cp\u003eThe result presented by the Helge H10 reader is binary and is communicated through a red or green light.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAn accessory screen, Zafena POC-Workstation (HemCheck Sweden AB), with integrated printer and barcode reader, was connected to the system to enable extraction of quantified free hemoglobin values, expressed in in mg/dL, g/L o \u0026mu;mol/L. The aim is to find hemolyzed blood samples in a few seconds in connection with the blood-sampling or quantifying the amount of free hemoglobin in the blood.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe innovative aspect is the detection of hemolysis in whole blood in REAL TIME, helping to ensure the quality of samples and to make immediate decisions.\u003c/p\u003e\n\u003cp\u003eA study at Swedish emergency department showed that a cut-off of 50 mg/ml PfHb has a sensitivity of 80%, specificity of 99%; positive and negative predictive values was 89% and 98% respectively. (7)\u003c/p\u003e\n\u003cp\u003eThe binary result of the reader is set with this cut off, but it could be changed.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eSTUDY DESIGN\u003c/h3\u003e\n\u003cp\u003eThe primary objective of this study was to demonstrate Helge\u003csup\u003eTM\u003c/sup\u003e V-Test effectiveness and safety in the early identification of hemolysis in patients undergoing elective cardiac surgery in ECC.\u003c/p\u003e\n\u003cp\u003eInclusion criteria were: indication for elective cardiac surgery in ECC, age \u0026ge; 18 years, ASA I-IV, informed consent approval; exclusion criteria were: not-approval informed consent, age \u0026lt; 18 years, ASA IVE, weight \u0026lt; 30 kg.\u003c/p\u003e\n\u003cp\u003eAll surgery were conducted under general anesthesia and ultrasound-guided bilateral parasternal block, with complete monitoring of vital parameters.\u003c/p\u003e\n\u003cp\u003eBlood samples were taken during the surgery at different times: T0: pre-ECC, T1: at aortic clamping, T2: after 20 minutes since the ECC started, T3: at the end of ECC, T4: at the end of surgery.\u003c/p\u003e\n\u003cp\u003eAll blood samples were performed from arterial access.\u003c/p\u003e\n\u003cp\u003eIn order to avoid handling errors and variations, a double sampling was carried out and both was tested. The blood samples were then analyzed using the Helge\u003csup\u003eTM\u003c/sup\u003e V-TEST disposable device by appropriately trained specialist anesthetists and residents. A total of ten test per patients were executed and collected. The cut-off to define the presence or absence of hemolysis was maintained at 50 mg/dL, as set by the manufacturer. (7)\u003c/p\u003e\n\u003cp\u003eFurthermore, patients\u0026apos; personal and anthropometric data were collected, as well as the pre- and post-operative hematocrit, the type of cannulas used by the perfusionists, the duration of the ECC and the clamping, the possible performance of pre-operative bloodletting and, finally, the use of vasoconstrictors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1721218001.png\"\u003e\u003c/p\u003e\n\u003ch3\u003eSTATISTICAL METHODS\u003c/h3\u003e\n\u003cp\u003eAll collected data were analyzed using the IBM SPSS Statistics editor and expressed in medians \u0026plusmn; \u0026nbsp;interquartile ranges.\u003c/p\u003e\n\u003cp\u003eThe data obtained from the two blood samples analyzed for each T were considered as the average of the two values (\u0026plusmn; standard deviation). The 1-Sample Wilcoxon test and the independent-samples Mann-Whitney test were performed on all T time values. The Spearman Rank test was used as a correlation test. Binary logistic regression was finally conducted to analyze the relationship between ECC and aortic clamping times and PfHb values at T1, T2 and T3 based on the data obtained from the rank test. P value was defined as \u0026lt; 0.05. The PfHb value is expressed in mg/dL.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eSince January 2022 to March 2023, 100 consecutive patients undergoing elective cardiac surgery in ECC were enrolled in the study. Informed consent was obtained from all of them. 18 were excluded for ASA IVE class risk and 4 were lost during data collecting. 78 patients were finally analyzed. (\u003cem\u003eTable 1\u003c/em\u003e)\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1721218523.png\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eOf these 78 patients, the median age was 71 (IR 33-89) years, 63 patients enrolled were women while 15 were men.\u003c/p\u003e\n\u003cp\u003eThe median body surface area (BSA) was 1.89 (IR 1,4-2,3) m\u003csup\u003e2\u003c/sup\u003e. The median preoperative hematocrit (HCT) was 40 (IR 25-51) % and the post-operative HCT was 30 (IR 22-42) %.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, the median duration of CEC was 70 (IR 30-193) min, while that of aortic clamping was 56 (IR 23-133). The median size of cannulae used in CEC was 32 (IR 15-40) Fr for arterials and 40 (IR 19-46) for venous one. (\u003cem\u003eTable 2\u003c/em\u003e)\u003c/p\u003e\n\u003cp\u003eOut of all cases analyzed, no case of hemolysis (PfHb value \u0026ge; 50 mg/dL) was recorded at T0. The frequency of hemolysis at T1 was 6.4% (5 of 78), at T2 was 5.1% (4 of 78), at T3 was 24.4% (19 of 78) and at T4 was 16.7% (13 of 78).\u003c/p\u003e\n\u003cp\u003eThe results recorded median PfHb values at T0 0.5 (IR 0-7.1) mg/dL, at T1 3.75 (IR 0-14.4) mg/dL; at T2 8.25 (IR 0.4-19.1) mg/dL, at T3 27.5 (IR 9.9-50) mg/dL, at T4 18.5 (IR 2.4-41) mg/dL; for all T time p value was \u0026lt; 0.001. (\u003cem\u003eTable 3-4\u003c/em\u003e)\u003c/p\u003e\n\u003cp\u003eTable 3 \u003cstrong\u003eFrequency of PfHb\u0026gt;50 mg/dL in all T times\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1721235657.png\"\u003e\u003c/strong\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eN. Absolute value; \u003cstrong\u003e%\u003c/strong\u003e Percentage value\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Table 4 \u003cstrong\u003ePfHb value in mg/dL in all T times\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1721235656.png\"\u003e\u003c/strong\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eA statistically significant correlation (p value \u0026lt; 0.001) was also found between the duration of the ECC and the PfHb value at T3 (end of ECC) and T4 (end of surgery). Likewise, the duration of aortic clamping was significantly correlated with the PfHb value at T3 and T4.\u003c/p\u003e\n\u003cp\u003eInstead, the PfHb value at all T time points was not related to patient age, gender, preoperative HCT value and ECC cannula diameter.\u003c/p\u003e\n\u003cp\u003eHemolysis occurred for a minimum ECC time of 35 minutes and a minimum clamp time of 31 minutes\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, our statistical analysis did not demonstrate a significant difference between PfHb values at T1 and at T2 (p = 0.130), a statistically significant difference was instead found between PfHb at all the other T value, particularly among PfHb values at T1-T2 and at T3 (p \u0026lt; 0.001). Complete data are shown in the table. (\u003cem\u003eTable 5\u003c/em\u003e)\u003c/p\u003e\n\u003cp\u003eBased on these data, we conducted a binary logistic regression analysis to correlate the occurrence of \u0026nbsp;hemolysis in T1, T2, and T3 and the duration of both ECC and aortic clamping.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1721218188.png\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eThese analyses demonstrated that there is no correlation between the duration of ECC and PfHB \u0026gt; 50 mg/dL in T1 and T2, whereas in T3 the probability of hemolysis increases for a ECC duration greater than 100 minutes. Similarly, the duration of aortic clamping does not influence the PfHb value at T1 and T2, but in T3 these increase if the clamping time is greater than 84 minutes. (\u003cem\u003eFigure 1\u003c/em\u003e)\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHemolysis in all extracorporeal circuits is a matter of fact.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThree distinct mechanisms are involved in hemolysis: natural selection in the spleen; physical or chemical imbalances, mechanical trauma. (8)\u003c/p\u003e\n\u003cp\u003eThe use of ECC in cardiac surgery determines the destruction of red blood cells throughout different pathways, with an increase in PfHb and a reduction in haptoglobin levels in the blood circulation. (3)\u003c/p\u003e\n\u003cp\u003eIn 2009, Pohlman and colleagues conducted a study on sheep and human blood samples reporting how the negative pressure exerted by the pump, together with the exposure of the blood to air, increased the degree of hemolysis. (9) Five years earlier Kameneva et al. reported how the intensity of hemolysis depends on the aspiration flow rate, the hematocrit, the duration of the ECC and the storage time of the concentrated red blood cells used during the CPB procedure.\u0026nbsp;(10)\u003c/p\u003e\n\u003cp\u003eOther authors suggest that the onset of hemolysis and inflammation also depends on the duration and conduction of CPB and on the material used in that process. (11)\u003c/p\u003e\n\u003cp\u003eThe release of free constituents of red blood cells and the consumption of scavenger mechanisms, as well as hemorheological alterations, lead to important clinical alterations that can promote organ failure.\u0026nbsp;(5)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe mechanisms of cellular mechanical damage during CPB have been described by several investigators: positive pressure, wall impact forces, nonendothelial blood surfaces, negative pressure, blood-air interface, and shear stress have all been identified as responsible forces.\u0026nbsp;(5)\u003c/p\u003e\n\u003cp\u003eIt is known that the extracorporeal circuit is made up of two different types of pumps: roller or centrifugal. If roller pumps force the blood flow inside a tube which is pinched by rotating rollers, centrifugal pumps move the flow by generating kinetic energy from the rotation of an impeller.\u0026nbsp;(12)\u003c/p\u003e\n\u003cp\u003eThe type of pump used to perform CPB can influence hemolysis; specifically, roller pumps are associated with a greater amount of hemolysis than centrifugal pumps. (13)\u003c/p\u003e\n\u003cp\u003eBhirowo and colleagues conducted a meta-analysis of 64 RCTs from 1990 to 2021 involving a total of 3384 patients undergoing various cardiac surgeries.\u003c/p\u003e\n\u003cp\u003eAccording to the authors, centrifugal pumps are associated with a significant reduction in hemolysis in terms of PfHb and HP levels compared to roller pumps (RP), but not in LDH levels. As in our trial, higher serum PfHb concentrations were reported in groups undergoing longer-duration ECC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, shear stress due to air aspiration via cardiotomy is considered another of the main cause of hemolysis during CPB. Significant reductions in hemolysis levels were observed in gravity-assisted venous drainage (GAVD) versus vacuum-assisted venous drainage (VAVD), continuous flow versus pulsatile flow, and using propofol versus isoflurane 1-2 %. (14)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Our study aims to demonstrate the effectiveness, speed and safety of the Helge\u003csup\u003eTM\u003c/sup\u003e-V-TEST device in identifying hemolysis in patients undergoing these interventions.\u003c/p\u003e\n\u003cp\u003eOur statistical analysis demonstrated a significant difference between PfHb values in T3 (end of ECC) and in T1-T2. The same difference is not present between T1 (aortic clamping) and T2 (20\u0026apos; after ECC started), therefore we can deduce that the only factor determining hemolysis is the duration of the ECC and the aortic clamping, given the same patient-related factors. In particular, the degree of hemolysis increases for ECCs exceeding 100 minutes.\u003c/p\u003e\n\u003cp\u003eStatistically significant differences were also found between T1-T2 and T0 (pre-ECC); however, the onset of ECC is an independent and non-modifiable factor for this type of surgery, unlike the duration.\u003c/p\u003e\n\u003cp\u003eTherefore, we can identify the cut off of 100 minutes of ECC beyond which it becomes justifiable to evaluate the degree of hemolysis with the Helge\u003csup\u003eTM\u003c/sup\u003e-V-TEST system to make intraoperative adjustments, in order to reduce the degree of hemolysis and its clinical consequences.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;For example, in patients in whom long-lasting ECC is expected it could be useful, as suggested by Vercaemst et al., the use of wider and shorter connecting tubes, avoiding angled bends, to minimize the gradients necessary to push the blood along the tube and the transition from laminar flow to turbulent flow. (5)\u003c/p\u003e\n\u003cp\u003eAt the same time, an increase in pfHb during ECC above the critical value of 50 mg/dL allows for the implementation of preventive strategies to avois clinically evident hemolysis, such as a reduction in hematocrit through hemodilution implemented directly by the perfusionist via CBP or, for adequate DO2 values, a reduction in ECC flows. Another strategy could be to switch from pulsed flow to continuous flow.\u003c/p\u003e\n\u003cp\u003eSimilarly, drugs such as pentoxifylin (PTX), polyethylene glycol (PEG) molecules, melatonin, simvastatin or inhaled NO (iNO), may have some benefits on hemolysis. (5)\u003c/p\u003e\n\u003cp\u003ePassaroni et al. studied the correlations between hemolysis and inflammation after ECC, with increased risk of organ dysfunction and multisystem complications, which could have a critical role on the duration of mechanical ventilation and post-operative stay in intensive care.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAcute renal failure (AKI) is a frequent complication after cardiac surgery in CPB and in 1-5% requires post-operative hemodialysis, with a significant increase in mortality and morbidity. (15)\u003c/p\u003e\n\u003cp\u003eThe causes are different and not all treatable, including age, anemia, diabetes, chronic lung disease, chronic heart failure, chronic renal dysfunction, administration of nephrotoxic agents, hypoperfusion, hypotension, embolization and increased aortic clamping time. A frequent cause is hemolysis.\u003c/p\u003e\n\u003cp\u003eHokka et al. conducted a prospective observational study on 74 patients with chronic renal failure who underwent cardiac surgery on the valve and aortic arch in ECC from 2014 to 2020, to evaluate the association between perioperative levels of free hemoglobin and haptoglobin and acute renal failure.\u003c/p\u003e\n\u003cp\u003eAKI occurred in 25 patients (33.8%) and their multivariable analysis demonstrated an independent increase in risk with maximum PfHb values and minimum haptoglobin values. The independent association of increased PfHb values with the risk of AKI occurred specifically one hour after the start of CPB.\u003c/p\u003e\n\u003cp\u003eThe authors suggest the use of filters to remove PfHb or the administration of exogenous haptoglobin or epicidin for the prevention of AKI in case of serum PfHb \u0026ge; 0.06 g/dL one hour after starting CPB or \u0026ge; 0 .12 g/dL at any time. (16)\u003c/p\u003e\n\u003cp\u003eAnother study evaluated the impact of hemolysis in cardiac surgery patients on plasma NO consumption, AKI and intestinal tissue damage after cardiac surgery. Likewise, in patients undergoing CPB, an increase in plasma PfHb levels and a reduction in plasma haptoglobin and, not surprisingly, in NO levels were recorded, in a manner proportional to the duration of ECC. These data were associated with an increase in urinary N-acetyl-b-D-glucosaminidase (NAG) and plasma intestinal fatty acid binding protein (IFABP), concluding that hemolysis plays an important role in postoperative renal damage and to the intestinal mucosa, as well as direct cytotoxic damage potentially limiting the bioavailability of NO. It is obvious that this has important consequences on the post-operative course, increasing the risk of AKI, but also of systemic inflammatory response (SIRS) or sepsis. (17)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll studies analyzed assessed hemolysis by measuring levels of PfHb, haptoglobin, LDH or by calculating the hemolysis index. (18)\u003c/p\u003e\n\u003cp\u003eA wide range of first- and second-line laboratory tests are currently available in the diagnostic approach to hemolysis but none of them can be performed bedside.\u003c/p\u003e\n\u003cp\u003eErythrocytes also contain the enzyme LDH, whose serum levels increase in case of hemolysis, but it cannot be considered a specific marker.\u003c/p\u003e\n\u003cp\u003eIn case of non-clinically evident hemolysis, once normocytic or, in some cases, macrocytic anemia has been identified, laboratory tests for a correct diagnosis should also include the measurement of reticulocytes and unconjugated bilirubin, which does not can no longer be eliminated properly, as can the search for blood in the urine. It is obvious that this request is an enormous waste of resources and above all of time.\u0026nbsp;(19)\u003c/p\u003e\n\u003cp\u003eThe laboratory measurement of hemiglobincyanide is currently the gold standard, but it is associated with toxicity and does not allow a timely diagnosis, on the other hand the evaluation of haptoglobin is associated with a high rate of false negatives. This has led to the development of new clinical chemistry analyzers in laboratories, such as the HIL index (Hemoglobin, icterus, lipaemia/turbidity) which can estimate the presence of fHb, bilirubin and turbidity in samples. (7)(18)\u003c/p\u003e\n\u003cp\u003eThe main strength of our study is the inclusion of patients undergoing different types of cardiac surgery of varying complexity. This allowed us to evaluate the effectiveness and safety of the Helge\u003csup\u003eTM\u003c/sup\u003e-V-TEST in different operating scenarios. Having established that the development of hemolysis is directly related to the extracorporeal circuit and the duration of ECC, implementing preventive interventions could help to limit hemolysis-related damage, to correct intraoperative management in case of PfHb rising and improve the post-operative outcome of these patients. Our study, however, was limited to evaluating the feasibility, reliability, and safety of the point-of-care examination, without distinction based on the type of surgery and patient comorbidities. For example, because haptoglobin is synthesized in the liver, patients with impaired liver function may have reduced haptoglobin production which could result in increased PfHb independent of red blood cell lysis. \u003cstrong\u003e(5)\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFrom a future perspective, it could be interesting to analyze the effectiveness of the test in the various subgroups of patients.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eEvaluating and quantifying hemolysis during ECC using a point-of-care system as Helge\u003csup\u003eTM\u003c/sup\u003e V-TEST allows real-time corrective strategies to be implemented to prevent hemolysis-related complications and could improve the outcome of patients undergoing elective cardiac surgery, especially for ECC duration over 100 minutes.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eThe authors have no relevant financial or non-financial interests to disclose.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eL.S. conceptualization, investigation, methodology, writing-review and editing.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eM.S. writing-original draft, formal analysis and data curation, software.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eA.M. writing-review and editing, validation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eF.L.V. conceptualization, investigation, methodology\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eA.S., A.L.D.P, D.S., S.R., M.C.C., L.M. data curation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eR.C. project administration and resources\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eF.C., G.P. supervision\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eA.R., E.C. conceptualization and data curation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eF.E.A., M.C. supervision, validation and visualization\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAll authors read and approved the final manuscript.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e\u003cstrong\u003eCorrons JLV, Casafont LB, Frasnedo EF (2021) \u003c/strong\u003e\u003cem\u003eConcise review: how do red blood cells born, live, and die? \u003c/em\u003eAnn Hematol. 100(10):2425-2433. https://doi.org/10.1007/s00277-021-04575-z\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eYoshida T, Prudent M, D\u0026apos;Alessandro A (2019)\u003c/strong\u003e \u003cem\u003eRed blood cell storage lesion: causes and potential clinical consequences.\u003c/em\u003e Blood Transfus. 17(1):27-52. https://doi.org/10.2450/2019.0217-18\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eVieira Junior FU, Antunes N, Vieira RW, \u0026Aacute;lvares LM, Costa ET (2012)\u003c/strong\u003e \u003cem\u003eHemolysis in extracorporeal circulation: relationship between time and procedures. \u003c/em\u003e Rev Bras Cir Cardiovasc. 27(4)535-541. https://doi.org/10.5935/1678-9741.20120095\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eShih AW, McFarlane A, Verhovsek M (2014)\u003c/strong\u003e \u003cem\u003eHaptoglobin testing in hemolysis: Measurement and interpretation. \u003c/em\u003eAm J Hematol. 89(4):443-447. https://doi.org/10.1002/ajh.23623\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eVercaemst L (2008)\u003c/strong\u003e \u003cem\u003eHemolysis in Cardiac Surgery Patients Undergoing Cardiopulmonary Bypass: A Review in Search of a Treatment Algorithm.\u003c/em\u003e J Extra Corpor Technol. 40(4):257-267\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eNemeto S, Aoki M, Dehua C, Imai Y (2000)\u003c/strong\u003e \u003cem\u003eFree Hemoglobin Impairs Cardiac Function in Neonatal Rabbit Hearts\u003c/em\u003e. Ann Thorac Surg. 69(5):1484-1489. https://doi.org/10.1016/s0003-4975(00)01176-0\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eDuhaldea H, Skogo J, Karlssonc M (2019)\u003c/strong\u003e \u003cem\u003ePoint-of-care hemolysis detection in blood gas specimens directly at the emergency department. \u003c/em\u003eScand J Clin Lab Invest. 79(5):283-287. https://doi.org/10.1080/00365513.2019.1612089\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003ePassaroni AC, Felicio ML, Campos NLKL, Silvia MAM, Yoshida WB (2018)\u003c/strong\u003e \u003cem\u003eHemolysis and Inflammatory Response to Extracorporeal Circulation during On-Pump CABG: Comparison between Roller and Centrifugal Pump Systems. \u003c/em\u003eBraz J Cardiovasc Surg. 33(1):64-71. https://doi.org/10.21470/1678-9741-2017-0125\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003ePohlmann JR, Toomasian JM, Hampton CE, Cook KE, Annich GM, Bartlett RH (2009) \u003c/strong\u003e\u003cem\u003eThe Relationships Between Air Exposure, Negative Pressure, and Hemolysis. \u003c/em\u003eASAIO J. 55(5):469-473. https://doi.org/10.1097/MAT.0b013e3181b28a5a\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eKameneva MV, Burgreen GW, Kono K, Repko B, Antaki JF, Umezu M (2004) \u003c/strong\u003e\u003cem\u003eEffects of Turbulent Stresses upon Mechanical Hemolysis: Experimental and Computational Analysis. \u003c/em\u003eASAIO J. 50(5):418-423. https://doi.org/10.1097/01.mat.0000136512.36370b5\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eMota AL, Rodrigues AJ, Evora PR (2008)\u003c/strong\u003e \u003cem\u003e[Adult cardiopulmonary bypass in the twenty-first century. Science, art or empiricism?]. \u003c/em\u003eRev Bras Cir Cardiovasc. 23(1):78-92. https://doi.org/10.1590/s0102-76382008000100013\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eGovender K, Jani VP, Cabrales P (2022)\u003c/strong\u003e \u003cem\u003eThe Disconnect Between Extracorporeal Circulation and the Microcirculation: A Review. \u003c/em\u003eASAIO J. 68(7):881-889. https://doi.org/10.1097/MAT.0000000000001618\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003ePaparella D, Galeone A, Venneri MT, Coviello M, Visicchio G, Cappabianca G, Maselli G, Marraudino N, Quaranta M, De Luca Tupputi Schinosa L. (2004)\u003c/strong\u003e \u003cem\u003eBlood damage related to cardiopulmonary bypass: in vivo and in vitro comparison of two different centrifugal pumps. \u003c/em\u003eASAIO J. 50(5):473-8. https://doi.org/10.1097/01.mat.0000136514.53139.d0\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eBhirowo YP, Raksawardana YK, Setianto BY, Sudadi S, Tandean TN, Zaharo AF, Ramsi IF, Kusumawardani HT, Triyono T. (2023)\u003c/strong\u003e \u003cem\u003eHemolysis and cardiopulmonary bypass: meta-analysis and systematic review of contributing factors.\u003c/em\u003e J Cardiothorac Surg. 18(1):291. https://doi.org/10.1186/s13019-023-02406-y.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eConlon PJ, Stafford-Smith M, White WD, Newman MF, King S, Winn MP, Landolfo K.\u003c/strong\u003e \u003cstrong\u003e(1999)\u003c/strong\u003e \u003cem\u003eAcute renal failure following cardiac surgery.\u003c/em\u003e Nephrol Dial Transplant. 14(5):1158-62. https://doi.org/10.1093/ndt/14.5.1158.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eHokka M, Egi M, Kubota K, Mizobuchi S.\u003c/strong\u003e \u003cstrong\u003e(2021)\u003c/strong\u003e \u003cem\u003ePerioperative Serum Free Hemoglobin and Haptoglobin Levels in Valvular and Aortic Surgery With Cardiopulmonary Bypass: Their Associations With Postoperative Kidney Injury.\u003c/em\u003e J Cardiothorac Vasc Anesth. 35(11):3207-3214. https://doi.org/10.1053/j.jvca.2021.04.029.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eVermeulen Windsant IC, de Wit NC, Sertorio JT, van Bijnen AA, Ganushchak YM, Heijmans JH, Tanus-Santos JE, Jacobs MJ, Maessen JG, Buurman WA. (2014)\u003c/strong\u003e \u003cem\u003eHemolysis during cardiac surgery is associated with increased intravascular nitric oxide consumption and perioperative kidney and intestinal tissue damage. \u003c/em\u003eFront Physiol. 5:340. https://doi.org/10.3389/fphys.2014.00340.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eLippi G, Favaloro EJ, Franchini M. Haemolysis index for the screening of intravascular haemolysis: a novel diagnostic opportunity?(2018)\u003c/strong\u003e \u003cem\u003eBlood Transfus. \u003c/em\u003e16(5):433-437. https://doi.org/10.2450/2018.0045-18.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003ePhillips J, Henderson AC. (2018) \u003c/strong\u003e\u003cem\u003eHemolytic Anemia: Evaluation and Differential Diagnosis. \u003c/em\u003eAm Fam Physician. 98(6):354-361. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4632556/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4632556/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eHemolysis is a complication in all surgical procedures requiring extracorporeal circulation (ECC). The aim of this study is to evaluate the effectiveness of the disposable point of care device Hemcheck Helge\u0026trade; V-TEST quantifying hemolysis during cardiac surgery in ECC.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTwo whole blood samples of 78 patients were simultaneously collected at different times of surgery: T0 pre ECC; T1 at the time of clamping of aorta; T2 at 20 minutes after the start of ECC; T3 at the end of ECC; T4 at the end of surgery. Both samples were analyzed by the disposable Hemcheck Helge\u0026trade; V-TEST device, which offers a real-time assessment of hemolysis through a value of plasma free hemoglobin (PfHb) expressed in mg/dL.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOut of all cases analyzed, no case of hemolysis (PfHb\u0026thinsp;\u0026ge;\u0026thinsp;50 mg/dL) was recorded at T0. The results recorded median PfHb values at T0 0.5 (0-7.1) mg/dL, at T1 3.75 (0-14.4) mg/dL; at T2 8.25 (0.4\u0026ndash;19.1) mg/dL, at T3 27.5 (9.9\u0026ndash;50) mg/dL, at T4 18.5 (2.4\u0026ndash;41) mg/dL; for all T time p value was \u0026lt;\u0026thinsp;0.001. A statistically significant correlation was recorded between hemolysis values\u0026thinsp;\u0026gt;\u0026thinsp;50 mg / dL at T3 and ECC time\u0026thinsp;\u0026gt;\u0026thinsp;100 minutes (p\u0026thinsp;\u0026lt;\u0026thinsp;0,05).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe use of Hemcheck Helge\u0026trade; V-TEST allows effective identification of hemolysis directly in the operating room, reducing wasted time for laboratory analyses. This could help the anesthetist, perfusionist or cardiac surgeon approaching earlier intraoperative hemolysis and its effects on organs function and improve the post-operative course of patients undergoing cardiac surgery in ECC.\u003c/p\u003e","manuscriptTitle":"Hemolysis intraoperative monitoring by Helge V-test in patients undergoing extracorporeal circulation in cardiac surgery: a single center data","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-19 01:39:15","doi":"10.21203/rs.3.rs-4632556/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3aecb453-5cc7-40f1-9896-86105cd2f406","owner":[],"postedDate":"July 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-11T09:14:15+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-19 01:39:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4632556","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4632556","identity":"rs-4632556","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}